17,20(Z)-dehydro vitamin D analogs and their uses

ABSTRACT

This invention discloses 17,20(Z)-dehydro vitamin D analogs, and specifically 17(Z)-1α,25-dihydroxy-17(20)-dehydro-2-methylene-19-nor-vitamin D 3  and pharmaceutical uses therefor. This compound exhibits pronounced activity in arresting the proliferation of undifferentiated cells and inducing their differentiation to the monocyte thus evidencing use as an anti-cancer agent and for the treatment of skin diseases such as psoriasis as well as skin conditions such as wrinkles, slack skin, dry skin and insufficient sebum secretion. This compound also may be used to treat autoimmune disorders and inflammatory diseases in humans as well as renal osteodystrophy and obesity. This compound also has significant calcemic activity making it a therapeutic agent for the treatment or prophylaxis of osteoporosis, osteomalacia, renal osteodystrophy and hypoparathyroidism.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.11/283,090, filed Nov. 18, 2005, now U.S. Pat. No. 7,241,748.

BACKGROUND OF THE INVENTION

This invention relates to vitamin D compounds, and more particularly to17,20(Z)-dehydro vitamin D analogs and their pharmaceutical uses, andespecially17(Z)-1α,25-dihydroxy-17(20)-dehydro-2-methylene-19-norvitamin D₃, itsbiological activities, and its pharmaceutical uses.

The natural hormone, 1α,25-dihydroxyvitamin D₃ and its analog in theergosterol series, i.e. 1α,25-dihydroxyvitamin D₂ are known to be highlypotent regulators of calcium homeostasis in animals and humans, andtheir activity in cellular differentiation has also been established,Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Manystructural analogs of these metabolites have been prepared and tested,including 1α-hydroxyvitamin D₃, 1α-hydroxyvitamin D₂, various side chainhomologated vitamins and fluorinated analogs. Some of these compoundsexhibit an interesting separation of activities in cell differentiationand calcium regulation. This difference in activity may be useful in thetreatment of a variety of diseases such as renal osteodystrophy, vitaminD-resistant rickets, osteoporosis, psoriasis, and certain malignancies.

Another class of vitamin D analogs, i.e. the so called 19-nor-vitamin Dcompounds, is characterized by the replacement of the A-ring exocyclicmethylene group (carbon 19), typical of the vitamin D system, by twohydrogen atoms. Biological testing of such 19-nor-analogs (e.g.,1α,25-dihydroxy-19-nor-vitamin D₃) revealed a selective activity profilewith high potency in inducing cellular differentiation, and very lowcalcium mobilizing activity. Thus, these compounds are potentiallyuseful as therapeutic agents for the treatment of malignancies, or thetreatment of various skin disorders. Two different methods of synthesisof such 19-nor-vitamin D analogs have been described (Perlman et al.,Tetrahedron Lett. 31, 1823 (1990); Perlman et al., Tetrahedron Lett. 32,7663 (1991), and DeLuca et al., U.S. Pat. No. 5,086,191).

In U.S. Pat. No. 4,666,634, 2β-hydroxy and alkoxy (e.g., ED-71) analogsof 1α,25-dihydroxyvitamin D₃ have been described and examined by Chugaigroup as potential drugs for osteoporosis and as antitumor agents. Seealso Okano et al., Biochem. Biophys. Res. Commun. 163, 1444 (1989).Other 2-substituted (with hydroxyalkyl, e.g., ED-120, and fluoroalkylgroups) A-ring analogs of 1α,25-dihydroxyvitamin D₃ have also beenprepared and tested (Miyamoto et al., Chem. Pharm. Bull. 41, 1111(1993); Nishii et al., Osteoporosis Int. Suppl. 1, 190 (1993); Posner etal., J. Org. Chem. 59, 7855 (1994), and J. Org. Chem. 60, 4617 (1995)).

2-substituted analogs of 1α,25-dihydroxy-19-nor-vitamin D₃ have alsobeen synthesized, i.e. compounds substituted at 2-position with hydroxyor alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713), with 2-alkylgroups (DeLuca et al U.S. Pat. No. 5,945,410), and with 2-alkylidenegroups (DeLuca et al U.S. Pat. No. 5,843,928), which exhibit interestingand selective activity profiles. All these studies indicate that bindingsites in vitamin D receptors can accommodate different substituents atC-2 in the synthesized vitamin D analogs.

In a continuing effort to explore the 19-nor class of pharmacologicallyimportant vitamin D compounds, analogs which are characterized by thepresence of a methylene substituent at carbon 2 (C-2), a hydroxyl groupat carbon 1 (C-1), and a shortened side chain attached to carbon 20(C-20) have also been synthesized and tested.1α-hydroxy-2-methylene-19-nor-pregnacalciferol is described in U.S. Pat.No. 6,566,352 while 1α-hydroxy-2-methylene-19-nor-homopregnacalciferolis described in U.S. Pat. No. 6,579,861 and1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol is described inU.S. Pat. No. 6,627,622. All three of these compounds have relativelyhigh binding activity to vitamin D receptors and relatively high celldifferentiation activity, but little if any calcemic activity ascompared to 1α,25-dihydroxyvitamin D₃. Their biological activities makethese compounds excellent candidates for a variety of pharmaceuticaluses, as set forth in the '352, '861 and '622 patents.

SUMMARY OF THE INVENTION

The present invention is directed toward 17,20(Z)-dehydro vitamin Danalogs, and their pharmaceutical uses, and more specifically toward17(Z)-1α,25-dihydroxy-17(20)-dehydro-2-methylene-19-norvitamin D₃, theirbiological activity, and various pharmaceutical uses for thesecompounds.

Structurally these 17,20(Z)-dehydro-vitamin D analogs are characterizedby the general formula I shown below:

where Y₁ and Y₂, which may be the same or different, are each selectedfrom the group consisting of hydrogen and a hydroxy-protecting group,where R₁₁ and R₁₂ are each hydrogen or taken together are a methylenegroup, where R₆ and R₇, which may be the same or different, are eachselected from the group consisting of hydrogen, alkyl, hydroxyalkyl,fluoroalkyl, hydroxy and alkoxy, or R₆ and R₇ when taken together mayrepresent the group —(CH₂)_(x)— where x is an integer from 2 to 5, or R₆and R₇ when taken together may represent the group ═CR₈R₉ where R₈ andR₉, which may be the same or different, are each selected from the groupconsisting of hydrogen, alkyl, hydroxyalkyl, fluoroalkyl, hydroxy andalkoxy, or when taken together R₈ and R₉ may represent the group—(CH₂)_(x)— where x is an integer from 2 to 5, and where the group Rrepresents any of the typical side chains known for vitamin D typecompounds.

More specifically R can represent a saturated or unsaturated hydrocarbonradical of 1 to 35 carbons, that may be straight-chain, branched orcyclic and that may contain one or more additional substituents, such ashydroxy- or protected-hydroxy groups, fluoro, carbonyl, ester, epoxy,amino or other heteroatomic groups. Preferred side chains of this typeare represented by the structure below

where the side chain and 17-ene double bond is in the Z configurationand where Z in the above side chain structure is selected from Y, —OY,—CH₂OY, —C≡CY and —CH═CHY, where the double bond in the side chain mayhave the cis or trans geometry, and where Y is selected from hydrogen,methyl, —COR⁵ and a radical of the structure:

where m and n, independently, represent the integers from 0 to 5, whereR¹ is selected from hydrogen, deuterium, hydroxy, protected hydroxy,fluoro, trifluoromethyl, and C₁₋₅-alkyl, which may be straight chain orbranched and, optionally, bear a hydroxy or protected-hydroxysubstituent, and where each of R², R³, and R⁴, independently, isselected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyland C₁₋₅ alkyl, which may be straight-chain or branched, and optionally,bear a hydroxy or protected-hydroxy substituent, and where R¹ and R²,taken together, represent an oxo group, or an alkylidene group having ageneral formula C_(k)H_(2k)— where k is an integer, the group ═CR²R³, orthe group —(CH₂)_(p)—, where p is an integer from 2 to 5, and where R³and R⁴, taken together, represent an oxo group, or the group—(CH₂)_(q)—, where q is an integer from 2 to 5, and where R⁵ representshydrogen, hydroxy, protected hydroxy, or C₁₋₅ alkyl and wherein any ofthe CH-groups at positions 20, 22, or 23 in the side chain may bereplaced by a nitrogen atom, or where any of the groups —CH(CH₃)—,—(CH₂)_(m)—, —CR₁R₂— or —(CH₂)_(n)— at positions 20, 22, and 23,respectively, may be replaced by an oxygen or sulfur atom.

The preferred analog is17(Z)-1α,25-dihydroxy-17(20)-dehydro-2-methylene-19-norvitamin D₃ whichhas the following formula Ia:

The above compounds of formula I, especially formula Ia, exhibit adesired, and highly advantageous, pattern of biological activity. Thesecompounds are characterized by relatively high binding to vitamin Dreceptors, as well as relatively high intestinal calcium transportactivity, as compared to that of 1α,25-dihydroxyvitamin D₃, and theyalso exhibit relatively high activity in their ability to mobilizecalcium from bone, as compared to 1α,25-dihydroxyvitamin D₃. Hence,these compounds can be characterized as having relatively high calcemicactivity. Their preferential activity on intestinal calcium transportand calcium mobilizing activity allows the in vivo administration ofthese compounds for the treatment and prophylaxis of metabolic bonediseases. Because of their preferential calcemic activity on gut calciumtransport and on bone, these compounds would be preferred therapeuticagents for the treatment and prophylaxis of diseases such asosteoporosis, especially low bone turnover osteoporosis, steroid inducedosteoporosis, senile osteoporosis or postmenopausal osteoporosis, aswell as osteomalacia and renal osteodystrophy. These analogs havingrelatively high calcemic activity while being very active on celldifferentiation are also expected to be useful as a therapy to treathypoparathyroidism since they are effective to raise blood calciumlevels.

The compounds I, and particularly Ia, of the invention have also beendiscovered to be especially suited for treatment and prophylaxis ofhuman disorders which are characterized by an imbalance in the immunesystem, e.g. in autoimmune diseases, including multiple sclerosis,lupus, diabetes mellitus, host versus graft rejection, and rejection oforgan transplants; and additionally for the treatment of inflammatorydiseases, such as rheumatoid arthritis, asthma, and inflammatory boweldiseases such as celiac disease, ulcerative colitis and Crohn's disease.Acne, alopecia and hypertension are other conditions which may betreated with the compounds of the invention.

The above compounds I, and particularly Ia, are also characterized byrelatively high cell differentiation activity. Thus, these compoundsalso provide therapeutic agents for the treatment of psoriasis, or as ananti-cancer agent, especially against leukemia, colon cancer, breastcancer, skin cancer and prostate cancer. In addition, due to theirrelatively high cell differentiation activity, these compounds providetherapeutic agents for the treatment of various skin conditionsincluding wrinkles, lack of adequate dermal hydration, i.e. dry skin,lack of adequate skin firmness, i.e. slack skin, and insufficient sebumsecretion. Use of these compounds thus not only results in moisturizingof skin but also improves the barrier function of skin.

The compounds of the invention of formula I, and particularly formulaIa, are also useful in preventing or treating obesity, inhibitingadipocyte differentiations, inhibiting SCD-1 gene transcription, and/orreducing body fat in animal subjects. Therefore, in some embodiments, amethod of preventing or treating obesity, inhibiting adipocytedifferentiations, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal subject includes administering to the animalsubject, an effective amount of one or more of the compounds or apharmaceutical composition that includes one or more of the compounds offormula I, and in particular the compound of formula Ia. Administrationof one or more of the compounds or the pharmaceutical compositions tothe subject inhibits adipocyte differentiation, inhibits genetranscription, and/or reduces body fat in the animal subject.

One or more of the compounds may be present in a composition to treat orprevent the above-noted diseases and disorders in an amount from about0.01 μg/gm to about 1000 μg/gm of the composition, preferably from about0.1 μg/gm to about 500 μg/gm of the composition, and may be administeredtopically, transdermally, orally, rectally, nasally, sublingually, orparenterally in dosages of from about 0.01 μg/day to about 1000 μg/day,preferably from about 0.1 μg/day to about 500 μg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-5 illustrate various biological activities of17(Z)-1α,25-dihydroxy-17(20)-dehydro-2-methylene-19-norvitamin D₃,hereinafter referred to as “VIT-III,” as compared to the native hormone1α,25-dihydroxyvitamin D₃, hereinafter “1,25(OH)₂D₃.”

FIG. 1 is a graph illustrating the relative activity of VIT-III and1,25(OH)₂D₃ to compete for binding with [³H]-1,25-(OH)₂-D₃ to thefull-length recombinant rat vitamin D receptor;

FIG. 2 is a graph illustrating the percent HL-60 cell differentiation asa function of the concentration of VIT-III and 1,25(OH)₂D₃;

FIG. 3 is a graph illustrating the in vitro transcription activity of1,25(OH)₂D₃ as compared to VIT-III;

FIG. 4 is a bar graph illustrating the bone calcium mobilizationactivity of 1,25(OH)₂D₃ as compared to VIT-III; and

FIG. 5 is a bar graph illustrating the intestinal calcium transportactivity of 1,25(OH)₂D₃ as compared to VIT-III.

DETAILED DESCRIPTION OF THE INVENTION

The preparation of 17,20(Z)-dehydro vitamin D analogs having thestructure I can be accomplished by a common general method, i.e. thecondensation of a bicyclic Windaus-Grundmann type ketone II with theallylic phosphine oxide III to the corresponding 17,20(Z)-dehydrovitamin D analog IV followed by deprotection at C-1 and C-3 to provideI:

In the structures III and IV, groups Y₁ and Y₂ are hydroxy-protectinggroups, preferably t-butyldimethylsilyl, it being also understood thatany functionalities that might be sensitive, or that interfere with thecondensation reaction, be suitably protected as is well-known in theart. The process shown above represents an application of the convergentsynthesis concept, which has been applied effectively for thepreparation of vitamin D compounds [e.g. Lythgoe et al., J. Chem. Soc.Perkin Trans. I, 590 (1978); Lythgoe, Chem. Soc. Rev. 9, 449 (1983); Tohet al., J. Org. Chem. 48, 1414 (1983); Baggiolini et al., J. Org. Chem.51, 3098 (1986); Sardina et al,. J. Org. Chem. 51, 1264 (1986); J. Org.Chem. 51, 1269 (1986); DeLuca et al., U.S. Pat. No. 5,086,191; DeLuca etal., U.S. Pat. No. 5,536,713].

The hydrindanones of the general structure II are not known. They can beprepared by the method shown in the Schemes herein (see the preparationof compound VIT-III).

For the preparation of the required phosphine oxides of generalstructure III, a synthetic route has been developed starting from amethyl quinicate derivative which is easily obtained from commercial(IR,3R,4S,5R)-(−)-quinic acid as described by Perlman et al.,Tetrahedron Lett. 32, 7663 (1991) and DeLuca et al., U.S. Pat. No.5,086,191.

The overall process of the synthesis of compounds I and Ia isillustrated and described more completely in U.S. Pat. No. 5,843,928entitled “2-Alkylidene-19-Nor-Vitamin D Compounds” the specification ofwhich is specifically incorporated herein by reference.

Particularly preferred 17,20(Z)-dehydro vitamin D analogs are thoseencompassed by general formula I wherein carbon-2 on the A-ring issubstituted with an alkylidene group or an alkyl group, or arehydrolyzable slow release compounds (whether substituted at carbon-2 ornot substituted at carbon-2).

2-Alkylidene Compounds

Structurally these 2-alkylidene analogs are characterized by the generalformula V shown below:

where Y₁, Y₂, R₁₁, R₁₂ and Z are as previously defined herein, and R₈and R₉, which may be the same or different, are each selected from thegroup consisting of hydrogen, alkyl, hydroxyalkyl and fluoroalkyl, or,when taken together represent the group —(CH₂)_(x)— where x is aninteger from 2 to 5.

2-Alkyl Compounds

Structurally these 2-alkyl analogs are characterized by the generalformula VI shown below:

where Y₁, Y₂, R₁₁, R₁₂ and Z are as previously defined herein, and R₁₀is selected from the group consisting of alkyl, hydroxyalkyl andfluoroalkyl.

Slow Release Compounds

Modified vitamin D compounds that exhibit a desirable and highlyadvantageous pattern of biological activity in vivo, namely, the moregradual onset and more prolonged duration of activity, may also be usedherein.

Structurally, the key feature of the modified vitamin D compounds havingthese desirable biological attributes is that they are derivatives of17,20(Z)-dehydro-vitamin D analogs, in which a hydrolyzable group isattached to the hydroxy group at carbon 25 and, optionally, to any otherof the hydroxy groups present in the molecule. Depending on variousstructural factors—e.g. the type, size, structural complexity—of theattached group, these derivatives hydrolyze to the active17,20(Z)-dehydro-vitamin D analog, at different rates in vivo, thusproviding for the “slow release” of the biologically active vitamin Dcompound in the body.

The “slow release” in vivo activity profiles of such compounds can, ofcourse, be further modulated by the use of mixtures of derivatives orthe use of mixtures consisting of one or more vitamin D derivativetogether with underivatized vitamin D compounds.

It is important to stress that the critical structural feature of thevitamin derivatives identified above is the presence of a hydrolyzablegroup attached to the hydroxy group at carbon 25 of the molecule. Thepresence of a hydrolyzable group at that position imparts on theresulting derivatives the desirable “slow-release” biological activityprofile mentioned above. Other hydroxy functions occurring in themolecule (e.g. hydroxy functions at carbons 1 or 3) may be present asfree hydroxy groups, or one or more of them may also be derivatived witha hydrolyzable group.

The “hydrolyzable group” present in the above-mentioned derivatives ispreferably an acyl group, i.e. a group of the type Q¹CO—, where Q¹represents hydrogen or a hydrocarbon radical of from 1 to 18 carbonsthat may be straight chain, cyclic, branched, saturated or unsaturated.Thus, for example, the hydrocarbon radical may be a straight chain orbranched alkyl group, or a straight chain or branched alkenyl group withone or more double bonds, or it may be an optionally substitutedcycloalkyl or cycloalkenyl group, or an aromatic group, such assubstituted or unsubstituted phenyl, benzyl or naphthyl. Especiallypreferred acyl groups are alkanoyl or alkenoyl groups, of which sometypical examples are formyl, acetyl, propanoyl, hexanoyl, isobutyryl,2-butenoyl, palmitoyl or oleoyl. Another suitable type of hydrolyzablegroup is the hydrocarbyloxycarbonyl group, i.e. a group of the typeQ²-O—CO—, where Q² is a C₁ to C₁₈ hydrocarbon radical as defined above.Exemplary of such hydrocarbon radicals are methyl, ethyl, propyl, andhigher straight chain or branched alkyl and alkenyl radicals, as well asaromatic hydrocarbon radicals such as phenyl or benzoyl.

These modified vitamin D compounds are hydrolyzable in vivo to theactive analog over a period of time following administration, and as aconsequence regulate the in vivo availability of the active analog,thereby also modulating their activity profile in vivo. The term“activity profile” refers to the biological response over time ofvitamin D compounds. Individual modified compounds, or mixtures of suchcompounds, can be administered to “fine tune” a desired time course ofresponse.

As used herein the term “modified vitamin D compound” encompasses anyvitamin D compound in which one or more of the hydroxy functions presentin such a compound are modified by derivatization with a hydrolyzablegroup. A “hydrolyzable group” is a hydroxy-modifying group that can behydrolyzed in vivo, so as to regenerate the free hydroxy functions.

In the context of this disclosure, the term hydrolyzable grouppreferably includes acyl and hydrocarbyloxycarbonyl groups, i.e. groupsof the type Q¹CO— and Q²-O—CO, respectively, where Q¹ and Q² have themeaning defining earlier.

Structurally, the modified vitamin D compounds encompassed may berepresented by the formula VII shown below:

where Y₁, Y₂, R₁₁, R₁₂, R₆, R₇ and Z are as previously defined hereinwith respect to formula I with the exception that R⁵ in the side chainis —OY₃ and Y₃ is an acyl group or a hydrocarbyloxycarbonyl group, aspreviously defined herein.

Some specific examples of such modified vitamin D compounds include2-substituted derivatives such as:

1,3,25-Triacetates where Y₁═Y₂═Y₃ and is CH₃CO;

1,3,25-Trihexanoates where Y₁═Y₂═Y₃ and is CH₃(CH₂)₄CO;

1,3,25-Trinonanoates where Y₁═Y₂═Y₃ and is CH₃(CH₂)₇CO; and

25-Acetates where Y₁═Y₂ and is H and Y₃ is CH₃CO.

These compounds can be prepared by known methods. See for exampleWO97/11053 published Mar. 27, 1999, and the previous description herein.

17(Z)-1α,25-dihydroxy-17(20)-dehydro-2-methylene-19-nor-vitamin D₃(referred to herein as VIT-III) was synthesized and tested.Structurally, this 19-nor analog is characterized by the general formulaIa previously illustrated herein.

The preparation of17(Z)-1α,25-dihydroxy-17(20)-dehydro-2-methylene-19-nor-vitamin D₃having the structure Ia can be accomplished by the condensation of abicyclic Windaus-Grundmann type ketone IIa with the allylic phosphineoxide IIIa to the corresponding 17(20)-dehydro-vitamin D analog IVafollowed by deprotection at C-1 and C-3 to provide Ia:

In the structures IIIa and IVa, groups Y₁ and Y₂ are hydroxy-protectinggroups, preferably t-butyldimethylsilyl, it being also understood thatany functionalities that might be sensitive, or that interfere with thecondensation reaction, be suitably protected as is well-known in theart. The process shown above represents a specific application of theconvergent synthesis concept, which was referred to previously hereinand has been applied effectively for the preparation of vitamin Dcompounds

The hydrindanone of the general structure IIa is not known. It can beprepared by the method shown in the Schemes herein (see the preparationof compound VIT-III).

As used in the description and in the claims, the term“hydroxy-protecting group” signifies any group commonly used for thetemporary protection of hydroxy functions, such as for example,alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafterreferred to simply as “silyl” groups), and alkoxyalkyl groups.Alkoxycarbonyl protecting groups are alkyl-O—CO— groupings such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl,benzyloxycarbonyl or allyloxycarbonyl. The term “acyl” signifies analkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or acarboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, malonyl,succinyl, glutaryl group, or an aromatic acyl group such as benzoyl, ora halo, nitro or alkyl substituted benzoyl group. The word “alkyl” asused in the description or the claims, denotes a straight-chain orbranched alkyl radical of 1 to 10 carbons, in all its isomeric forms.“Alkoxy” refers to any alkyl radical which is attached by oxygen, i.e. agroup represented by “alkyl-o-.” Alkoxyalkyl protecting groups aregroupings such as methoxymethyl, ethoxymethyl, methoxyethoxymethyl, ortetrahydrofuranyl and tetrahydropyranyl. Preferred silyl-protectinggroups are trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,dibutylmethylsilyl, diphenylmethylsilyl, phenyldimethylsilyl,diphenyl-t-butylsilyl and analogous alkylated silyl radicals. The term“aryl” specifies a phenyl-, or an alkyl-, nitro- or halo-substitutedphenyl group.

A “protected hydroxy” group is a hydroxy group derivatised or protectedby any of the above groups commonly used for the temporary or permanentprotection of hydroxy functions, e.g. the silyl, alkoxyalkyl, acyl oralkoxycarbonyl groups, as previously defined. The terms “hydroxyalkyl”,“deuteroalkyl” and “fluoroalkyl” refer to an alkyl radical substitutedby one or more hydroxy, deuterium or fluoro groups respectively. An“alkylidene” refers to a radical having the general formula C_(k)H_(2k)—where k is an integer.

More specifically, reference should be made to the following descriptionas well as to the Schemes herein for a detailed illustration of thepreparation of compound VIT-III.

Synthesis

Des-A,B-23,24-dinorcholan-8β,22-diol (2). A flame dried 1000 mL twonecked flask was charged with ergocalciferol 1 (5 g, 12.6 mmol),pyridine (5 mL), and anhydrous MeOH (400 mL). The solution was cooled to−78° C. in an argon atmosphere. O₃ was bubbled through the solutionuntil a deep blue color developed and persisted (about 1 h). Thesolution was treated with O₂ until the blue color faded (15 min). ThenNaBH₄ (1.5 g, 39.7 mmol) was added. After 15 min. second portion ofNaBH₄ (1.5 g, 39.7 mmol) was added and the reaction was allowed to warmto rt. Then the third portion of NaBH₄ (1.5 g, 39.7 mmol) was added andreaction stirred for over night. The reaction was quenched by addingwater (50 mL). Methanol was evaporated in vaccuo and residue wasdissolved in ethyl acetate. The organic phase was washed with 1N aqueoussolution of HCl (100 mL), saturated NaHCO₃ solution (100 mL) and brine(100 mL). The organic phase was dried (Na₂SO₄), filtered and evaporated.Purification by silica gel chromatography (25% ethyl acetate/hexane)afforded 2.18 g (10.3 mmol, 81%) of diol 2 as a white solid. Mp 110-111°C.; ¹H NMR (400 MHz, CDCl₃) δ: 0.96 (3H, s), 1.03 (3H, d, J=6.6 Hz),3.38 (1H, dd, J=10.5, 6.7 Hz), 3.64 (1H, dd, J=10.5, 3.2 Hz), 4.09 (1H,m); ¹³C NMR (100 MHz, CDCl₃) δ: 69.2, 67.8, 52.9, 52.4, 41.8, 40.2,38.2, 33.6, 26.6, 22.6, 17.4, 16.6, 13.6; MS m/z (relative intensity):212 (M⁺, 2), 194 (M⁺-H₂O, 15), 179 (M⁺-H₂O—CH₃, 18), 125 (43), 111(100); exact mass calculated for C₁₃H₂₂O [M-H₂O]⁺ is 194.1671, measuredis 194.1665.

Des-A,B-22-(p-toluenesulfonyloxy)-23,24-dinorcholan-8β-ol (3). Asolution of diol 2 (1 g, 4.71 mmol) in anhydrous pyridine (12 mL) wascooled to −25° C. and a precooled solution of tosyl chloride (1.08 g,5.66 mmol) in anhydrous pyridine (2 mL) was added dropwise. The reactionmixture was stirred at that temperature for 4 h and allowed to warm to0° C. and stirred at that temperature for additional 20 h. The mixturewas diluted with CH₂Cl₂ (50 mL) and washed with saturated CuSO₄ solution(30 mL), 1N HCl (30 mL), and water (50 mL). The organic phase was dried(Na₂SO₄), filtered and concentrated. Purification by silica gelchromatography (25% ethyl acetate/hexane) yielded 1.7 g (4.64 mmol, 98%)of tosylate 3. ¹H NMR (400 MHz, CDCl₃) δ: 0.89 (3H, s), 0.96 (3H, d,J=6.6 Hz), 2.45 (3H, s), 3.8 (1H, dd, J=9.2, 6.2 Hz), 3.95 (1H, dd,J=9.2, 3.0 Hz), 4.06 (1H, m), 7.35 (2H, d, J=8.2 Hz), 7.78 (2H), d,J=8.2 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 144.7, 133.0, 129.8, 127.9, 75.6,69.0, 60.4, 52.2, 41.9, 40.1, 35.7, 33.5, 26.4, 22.4, 21.6, 17.3, 16.7,13.4; MS m/z (relative integration): 366 (M⁺, 6), 194(14), 179(16),125(30), 111(100); exact mass calculated for C₂₀H₃₀SO₄Na (M+Na⁺) is389.1763, measured is 389.1768.

Des-A,B-8β-[(tert-butyldimethylsilyl)oxy]-22-(p-toluenesulfonyloxy)-23,24-dinorcholane(4). To a 0° C. cooled solution of hydroxyl tosylate 3 (1.5 g, 4.09mmol) in anhydrous DMF (20 mL) was added 2,6-lutidine (0.580 mL, 0.52 g,4.92 mmol) followed by TBSOTf (1.13 mL, 1.30 g, 4.92 mmol). The solutionwas stirred at 0° C. for 15 min and water (10 mL) was added. The mixturewas extracted with ethyl acetate (3×40 mL), and combined organic phaseswere washed with 1N aqueous solution of NaOH (40 mL) dried (Na₂SO₄),filtered and concentrated. The residue was purified by silica gel columnchromatography (5% ethyl acetate/hexane) to give 1.94 g (4.04 mmol, 99%)of 4. ¹H NMR (400 MHz, CDCl₃) δ: 0.01 (6H, s), 0.88 (12H, s), 0.96 (3H,d, J=6.8 Hz), 2.45 (3H, s), 3.81 (1H, dd, J=9.2, 6.4 Hz), 3.97 (1H, dd,J=9.7, 3.0 Hz), 3.99 (1H, m), 7.34 (2H, d, J=8.08 Hz), 7.79 (2H, d,J=8.2 Hz). ¹³C NMR (100 MHz, CDCl₃) δ: 114.5, 133.4, 129.8, 127.9, 74.8,69.3, 52.3, 52.6, 42.2, 40.5, 35.8, 34.4, 26.6, 25.9, 23.0, 21.6, 18.0,17.6, 16.8, 13.7, −4.8, −5.1.

Des-A,B-8β-[(tert-butyldimethylsilyl)oxy]-23,24-dinorcholan-22-al (5). Asolution of 4 (1.9 g, 3.96 mmol) in DMSO (5 mL) was added to asuspension of NaHCO₃ (1.5 g, 17.9 mmol) in DMSO (20 mL) at rt. Themixture was heated to 150° C. under argon for 15 min and cooled to rt.Water (50 mL) followed by ethyl acetate (50 mL) were added and aqueousphase was extracted with ethyl acetate (3×30 mL). The combined organicphases were dried (Na₂SO₄), filtered and concentrated. The residue waspurified by column chromatography (2% ethyl acetate/hexane) to afford0.93 g (2.87 mmol, 76%) of aldehyde 5. H NMR (400 MHz, CDCl₃) δ: 0.01(6H, 2s), 0.89 (9H, s), 0.97 (3H, s), 1.09 (3H, d, J=6.8 Hz), 2.35 (1H,m), 4.03 (1H, m), 9.58 (1H, d, J=3.2 Hz). ¹³C NMR (100 MHz, CDCl₃) δ:205.2, 69.1, 52.4, 51.8, 49.1, 42.7, 40.5, 30.8, 34.3, 26.2, 25.8, 23.3,17.6, 14.1, 13.3, −4.7, −5.1.

Des-A,B-8β-[(tert-butyldimethylsilyl)oxy]-pregnan-20-one (6). A flamedried flask was charged with t-BuOK (1.55 g, 13.9 mmol) and anhydroust-BuOH (30 mL) at room temperature. O₂ was bubbled through the solutionfor 15 min. A solution of aldehyde 5 (0.9 g, 2.78 mmol) in anhydroust-BuOH (15 mL) was added to the reaction mixture and O₂ was bubbledthrough the solution for additional 10 min. The reaction was quenchedwith water (15 mL) and extracted with ether (3×30 mL). The combinedorganic phases were dried (Na₂SO₄), filtered and concentrated. Theresidue was purified by silica gel column chromatography (3% ethylacetate/hexane) to give 0.61 g (1.97 mmol, 71%) of the ketone 6. ¹H NMR(400 MHz, CDCl₃) δ: 0.01 (6H, s), 0.84 (3H, s), 0.87 (9H, s), 2.08 (3H,s), 2.46 (1H, t, J=9.1 Hz), 4.03 (1H, m). ¹³C NMR (100 MHz, CDCl₃) δ:209.5, 69.0, 64.5, 53.2, 43.7, 39.8, 34.2, 31.6, 25.8, 23.2, 21.8, 17.6,15.5, −4.8, −5.2.

5-Bromo-2-methyl-2-pentanol (8). To a −20° C. cooled solution ofethyl-4-bromobutyrate 7 (5 g, 25.6 mmol) in anhydrous diethyl ether (50mL) was added 3M solution of methylmagnesium bromide in diethyl ether(17.1 mL, 6.11 g, 51.3 mmol) under argon atmosphere over a period of 30min. The reaction mixture was stirred at room temperature for overnight.Saturated ammonium chloride solution was added to hydrolyze the reactionmixture followed by 1N HCl solution to dissolve the inorganic saltsformed. The aqueous phase was extracted with ether (3×50 mL). Thecombined extracts were washed with water (100 mL), saturated NaClsolution (100 mL), dried (Na₂SO₄), filtered and concentrated. Theresidue was purified by silica gel column chromatography (20/80 ethylacetate/hexane) to afford 3.1 g (17.1 mmol, 67%) of tertiary alcohol 8.¹H NMR (400 MHz, CDCl₃) δ: 1.27 (6H, s), 1.64 (2H, m), 1.96 (2H, m),3.44 (2H, t, J=6.68 Hz).

5-Bromo-2methyl-2[(tert-butyldimethylsilyl)oxy]-pentane (9). To a −50°C. cooled solution of alcohol 8 (3 g, 16.6 mmol) in anhydrous CH₂Cl₂ (50mL) was added 2,6-lutidine (2.32 mL, 2.13 g, 19.89 mmol) followed byTBSOTf (4.57 mL, 5.26 g, 19.9 mmol). The solution was stirred at 0° C.for 15 min and water (10 mL) was added. The mixture was extracted withCH₂Cl₂ (3×40 mL), and combined organic phases were washed with 1Naqueous solution of NaOH (40 mL), dried (Na₂SO₄), filtered andconcentrated. The residue was purified by silica gel columnchromatography (1% ethyl acetate/hexane) to give 3.9 g (13.2 mmol, 80%)of 9. ¹H NMR (400 MHz, CDCl₃) δ: 0.07 (6H, s), 0.85 (9H, s), 1.21 (6H,s), 1.55 (2H, m), 1.95 (2H, m), 3.41 (2H, t, J=6.8 Hz)

Des-A,B-cholest-17(20)-dehydro-8β,25-diols (15a and 15b)

A solution of 5-bromo-2methyl-2[(tert-butyldimethylsilyl)oxy]pentane 9(2.84 g, 9.68 mmol) in anhydrous ether (20 mL, containing catalyticamount of iodine) was added dropwise to a stirred suspension ofmagnesium powder (0.23 g, 9.68 mmol) in anhydrous diethyl ether (5 mL)at room temperature with occasional warming it up to 35° C. under argonatmosphere. After generation of the Grignard reagent was complete themixture was stirred for 1 hr at room temperature and for 1 hr at 40° C.Then it was cooled to 0° C. and a solution of ketone 6 (0.6 g, 1.94mmol) in anhydrous diethyl ether (10 mL) was added dropwise over aperiod of 30 min. After stirring the reaction mixture at roomtemperature for 3 h it was hydrolyzed with aqueous solution of NH₄Cl (20mL). The organic layer was separated and aqueous phase was extractedwith ethyl acetate (3×30 mL). The combined organic phases were washedwith water (40 mL), dried (Na₂SO₄) and evaporated. Silica gel columnchromatography of the residue gave 0.95 g (94%) of mixture of alcohols10. Phosphorous oxychloride (3 mL) was added dropwise to a solution ofmixture of alcohols 10 (0.95 g) in anhydrous pyridine (20 mL) underargon atmosphere. The reaction was stirred at room temperature overnightand poured into ice-water and extracted with ether (3×20 mL). Theorganic layer was washed with saturated CuSO₄ solution (30 mL), 1N HCl(30 mL), water (50 mL). The organic phase was dried (NaSO₄), filteredand concentrated. Column Chromatography of crude mixture furnished 0.72g (78%) of mixture of olefins 11a, 11b, 12a, 12b, 13. The olefin mixturewithout further purification was dissolved in methanol (20 mL) andp-Toluenesulfonic acid monohydrate (p-TSA) (0.100 g) was added at 0° C.The reaction mixture was stirred at room temperature for 3 days[Additional amounts of p-TSA were successively added (100 mg, 24 h; 75mg, 36 h; 50 mg, 48 h)]. Methanol was evaporated and residue was dilutedwith ethyl acetate (30 mL). The organic phase was washed with saturatedaqueous NaHCO₃ solution (20 mL) water (20 mL), dried (Na₂CO₃) andevaporated. The residue was purified on column chromatography to yield0.284 g (79%) of mixture of olefin alcohols 14a, 14b, 15a, 15b, 16. Theolefin alcohols were separated on HPLC.

17(E)-Des-A,B-cholestan-17(20)-dehydro-8β,25-diol (15a). The olefinalcohols were separated on HPLC (9.4 mm×25 cm zorbax-sil column, 4ml/min) using IPA/hexane (4/96) solvent system. Pure diol 17-20E 15a 70mg (250 μmol, 25%) was eluted at Rv=50 mL. [α]²⁵ _(D)−16.5° (c 1.02,CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ: 1.09 (3H, s), 1.20 (6H, s), 1.67 (3H,t, J=1.84 Hz), 4.14 (1H, m). ¹³C NMR (100 MHz, CDCl₃) δ: 143.2, 123.7,71.0, 69.8, 52.4, 43.9, 43.7, 38.3, 36.8, 33.4, 29.2, 28.5, 23.5, 22.2,19.1, 17.9, 17.2. MS m/z (relative intensity): 280 (M⁺, 16), 262(M-H₂O⁺, 7), 229 (M-2×H₂O—CH₃ ⁺, 16) 179(54), 161(100); Exact masscalculated for C₁₈H₃₂O₂ [M+Na]⁺ is 303.2300, found 303.2297.

17(E)-25-(Triethylsilyloxy)-des-A,B-cholestan-17(20)-dehydro-8-one(17a). To a solution of alcohol 15a (20 mg, 71 μmol) in anhydrous CH₂Cl₂(5 mL) was added PDC (40 mg, 107 μmol) at rt. After stirring thereaction for 3 h under argon atmosphere the solution was passed througha pad of celite with ethyl acetate. The filtrate was concentrated andapplied on a Sep-Pak cartridge and eluted with ethyl acetate/hexane(20/80) to give 17 mg, (61.1 μmol, 86%) of ketone as colorless oil. To a−50° C. cooled solution of ketone (17 mg, 61.1 μmol) in anhydrous CH₂Cl₂(5 mL) was added 2,6-lutidine (9 μL, 7.86 mg, 73.3 μmol) followed byTESOTf (17 μL, 19.4 mg, 73.3 μmol). The solution was stirred at 0° C.for 15 min and water (5 mL) was added. The mixture was extracted withCH₂Cl₂ (3×5 mL), and combined organic phases were dried (Na₂SO₄),filtered and concentrated. The ketone was purified on HPLC (9.4-mm×25-cmZorbax-Sil column, 4 ml/min) using 10% ethyl acetate/hexane solventsystem. Pure ketone 17a 14.4 mg (36.7 μmol, 60%) was eluted at R_(v)=20mL as colorless oil. [α]²⁵ _(D)-14.4 (c 0.73, CHCl₃); ¹H NMR (400 MHz,CDCl₃) δ: 0.56 (6H, q, J=7.7 Hz), 0.84 (3H, s), 0.94 (9H, t, J=4.76 Hz),1.18 (6H, s), 1.71 (3H, t, J=1.84 Hz), 2.57 (1H, dd, J=12, 6.2 Hz). ¹³CNMR (100 MHz, CDCl₃) δ: 212.2, 141.2, 126.1, 73.3, 61.8, 50.5, 44.7,40.6, 36.9, 36.7, 29.9, 29.8, 28.7, 23.9, 22.1, 20.2, 17.8, 17.6, 7.1,6.8. MS m/z (relative intensity): No M⁺, 377([M-CH₃]⁺, 3) 363([M-C₂H₅]⁺,9), 204(100), 189((18), 161(45). Exact mass calculated for C₂₄H₄₄O₂Si[M+Na]⁺ is 415.3008, found 415.3016.

17(E)-1α,25 Dihydroxy-17(20)-dehydro-2-methylene-19-norvitamin D₃ (20a).To a solution of phosphine oxide 18 (0.051 g, 87.6 μmol) in anhydrousTHF (500 μL) at −25° C. was slowly added PhLi 1.2M in cyclohexane/ether(70/30) (80 μL, 8.1 mg, 96.4 μmol) under argon with stirring. Thesolution turned deep orange. The mixture was stirred at that temperaturefor 20 min and cooled to −78° C. A precooled (−78° C.) solution ofketone 17a (14 mg, 35.7 μmol) in anhydrous THF (100 μL) was addedslowly. The mixture was stirred under argon atmosphere at −78° C. for 3h and at 0° C. for 18 h. Ethyl acetate was added and organic phase waswashed with brine, dried (Na₂SO₄) and evaporated. The residue wasapplied on a Sep-Pak cartridge, and eluted with 1% ethyl acetate/hexaneto give 19-nor protected vitamin derivative 19a (8 mg of unreactedketone 17a was recovered). The protected vitamin was further purified byHPLC (9.4-mm×25-cm Zorbax-Sil column, 4 ml/min) using hexane/IPA(99.95/0.05) solvent system. Pure compound 19a, 7.7 mg (10.2 μmol, 29%)was eluted at R_(v)=20 mL as colorless oil. UV (in hexane) λ_(max)243.1, 252, 262.2 nm; ¹H NMR (400 MHz, CDCl₃) δ: 0.03, 0.05, 0.07, 0.08(each 3H, each s), 0.56 (6H, q, J=7.8 Hz), 0.74 (3H, s), 0.87 and 0.91(each 9H, each s), 0.96 (9H, t, J=7.8 Hz), 1.19 (6H, s), 1.68 (3H, t,J=1.86 Hz), 2.18 (1H, dd, J=12.6, 8.3 Hz), 2.33 (1H, m) 2.46 (1H, dd,12.6, 4.6 Hz), 2.53 (1H, dd, 13.3, 5.88 Hz), 2.80 (1H, m), 4.43 (2H, m),4.93 and 4.97 (1H and 1H, each s), 5.88 and 6.21 (1H and 1H, each d,J=11.2 Hz); MS m/z (relative intensity): No M⁺, 624(59), 366(32),91(100); Exact mass calculated for C₄₅H₈₄O₃Si₃ [M+Na]⁻ is 779.5626,found 779.5648.

The protected vitamin 19a (7.7 mg, 10.2 μmol) was dissolved in anhydrousTHF (500 μL) and treated with TBAF (0.102 mL, 26.7 mg, 102 μmol) andstirred at rt in dark for overnight. The solvent was removed in vaccuoand residue was applied on Sep-Pak cartridge, and eluted with 30% ethylacetate/hexane to get the deprotected vitamin 20a. The vitamin wasfurther purified by HPLC (9.4-mm×25-cm Zorbax-Sil column, 3 mL/min)using hexane/IPA (90/10) as solvent system. Pure vitamin 20a, 2.9 mg (7μmol, 69%) was collected at R_(v)=42 mL as white solid: UV (in EtOH)λ_(max)242.9, 251, 261.2 nm; ¹H NMR (500 MHz, CDCl₃) δ: 0.74 (3H, s),1.22 (6H, s), 1.69 (3H, t, J=1.94 Hz,), 2.29 (1H, dd, J=13.0, 8.39 Hz),2.32 (1H, dd, J=13.9, 7.0 Hz), 2.57 (1H, dd, J=13.4, 3.49 Hz), 2.79 (1H,br d) 2.87 (1H, dd, J=13.0, 4.59 Hz), 4.49 (2H, m), 5.09 and 5.11 (1Hand 1H, each s), 5.92 and 6.35 (1H and 1H, each d, J=11.29 Hz); MS m/z(relative intensity): 414 (M⁺, 36), 396([M-H₂O]⁺, 6), 381([M-H₂O—CH₃]⁺,8) 285(70), 149(61), 69(100).

17(Z)-Des-A,B-cholest-17(20)-dehydro-8β,25-diol (15b). The olefinalcohols were separated on HPLC (9.4 mm×25 cm zorbax-sil column, 4ml/min) using IPA/hexane (5/95) solvent system. Diol 17-20Z 15b and Diol20-21 16 eluted out together at Rv=45 mL. The alcohols were oxidizedtogether.

17(Z)-25-(Triethylsilyloxy)-des-A,B-cholest-17(20)-dehydro-8-one (17b).To a solution of mixture of alcohols 15b and 16 (34 mg, 121 μmol) inanhydrous CH₂Cl₂ (5 mL) was added PDC (55 mg, 145.7 μmol) at rt. Afterstirring the reaction for 3 h under argon atmosphere the solution waspassed through a pad of celite with ethyl acetate. The filtrate wasconcentrated and applied on a Sep-Pak cartridge and eluted with ethylacetate/hexane (20/80) to give a mixture of ketones 17b and 16b 30.2 mg(108.6 μmol, 89%) as colorless oil. To a −50° C. cooled solution ofketones 30.2 mg (30.2 mg, 108.6 μmol) in anhydrous CH₂Cl₂ (10 mL) wasadded 2,6-lutidine (16 μL, 13.9 mg, 130.3 μmol) followed by TESOTf (30μL, 34.5 mg, 130.3 μmol). The solution was stirred at 0° C. for 15 minand water (10 mL) was added. The mixture was extracted with CH₂Cl₂ (3×5mL), and combined organic phases dried (Na₂SO₄), filtered andconcentrated. The residue was purified by HPLC (9.4-mm×25-cm Zorbax-Silcolumn, 4 ml/min) using ethyl acetate/hexane (5/95) solvent system. Pureketone 17b 7.7 mg (19.6 μmol, 18%) was eluted at R_(v)=34 mL ascolorless oil. ¹H NMR (400.13 MHz, CDCl₃) δ: 0.56 (6H, q, J=7.78 Hz),0.83 (3H, s), 0.94 (9H, t, J=7.9 Hz), 1.2 (6H, s), 1.57 (3H, br s), 2.57(1H, dd, J=11.8, 6.3 Hz); ¹³CNMR (100 MHz, CDCl₃) δ: 212.18, 141.1,126.8, 73.2, 62.0, 50.5, 45.3, 40.7, 37.1, 34.5, 29.9, 29.8, 24.0, 23.8,20.2, 20.1, 18.7, 7.1, 6.8. MS m/z (relative intensity): No M⁺, 363([M-C₂H₅]⁺, 10), 334 ([M-2×C₂H₅]⁺, 1), 204 (100).

17(Z)-1α,25 Dihydroxy-17(20)-dehydro-2-methylene-19-norvitamin D₃ (20b).To a solution of phosphine oxide 10 (62 mg, 106.5 μmol) in anhydrous THF(750 μL) at −25° C. was slowly added PhLi 1.8 M in Di-n-butyl ether (59μL, 8.9 mg, 106.5 μmol) under argon with stirring. The solution turneddeep orange. The mixture was stirred at that temperature for 20 min andcooled to −78° C. A precooled (−78° C.) solution of ketone 17b (7.7 mg,19.6 μmol) in anhydrous THF (100 μL) was added slowly. The mixture wasstirred under argon atmosphere at −78° C. for 3 h and at 0° C. for 18 h.Ethyl acetate was added and organic phase was washed with brine, dried(Na₂SO₄) and evaporated. The residue was applied on a Sep-Pak cartridge,and eluted with 1% ethyl acetate/hexane to give the 19-nor protectedvitamin derivative. The vitamin was further purified by HPLC(9.4-mm×25-cm Zorbax-Sil column, 4 ml/min) using hexane/IPA (99.95:0.05)solvent system. Pure compound 19b, 12.8 mg (16.9 μmol, 86%) was elutedat R_(v)=19 mL as colorless oil. [α]²⁵ _(D)−9.35 (c 0.64, CHCl₃); UV (inhexane): λ_(max)244.4, 253.2, 263.2 nm; ¹H NMR (400 MHz, CDCl₃) δ:0.026, 0.050, 0.067, 0.082 (each 3H, each s), 0.56 (6H, q, J=7.84 Hz),0.74 (3H, s), 0.86, 0.89 (each 9H, each s), 0.94 (9H, t, J=7.96 Hz),1.19 (6H, s), 1.56 (3H, br s), 2.14 (1H, dd, J=12.5, 4.8 Hz), 2.33 (1H,dd, J=13.1, 2.8 Hz), 2.46 (1H, dd, J=12.7, 4.4 Hz), 2.53 (1H, dd,J=13.3, 6.0 Hz), 2.80 (1H, br d, J=13.5 Hz), 4.43 (2H, m), 4.92 and 4.97(each 1H, each s), 5.88 and 6.21 (each 1H, each d, J=11.1 Hz); ¹³C NMR(100 MHz, CDCl₃) δ: 152.9, 142.2, 140.8, 132.8, 125.9, 122.3, 116.3,106.2, 73.3, 72.5, 71.6, 56.7, 47.6, 46.8, 45.4, 30.1, 29.8, 28.5, 25.8,25.7, 23.9, 23.6, 23.0, 19.9, 18.2, 18.1, 17.8, 7.1, 6.8, −4.8, −5.0; MSm/z (relative intensity): No M⁺, 366(2), 263(100); Exact mass calculatedfor C₄₅H₈₄O₃Si₃[M+Na]⁺ is 779.5624, found 779.5647.

The protected vitamin 19b (12.8 mg, 16.9 mmol) was dissolved inanhydrous THF (500 μL) and treated with TBAF (170 μL, 44.2 mg, 169 μmol)and stirred at rt in dark for overnight. The solvent was removed invaccuo and residue was applied on Sep-Pak cartridge, and eluted with 30%ethyl acetate/hexane to get the deprotected vitamin. The vitamin wasfurther purified by HPLC (9.4-mm×25-cm Zorbax-Sil column, 4 ml/min)using hexane/IPA (85/15) as solvent system. Pure vitamin 20b, 4.3 mg(10.3 μmol, 62%) was eluted at R_(v)=33 mL. UV (in ethanol):λ_(max)244.1, 252.5, 262.1 nm; ¹H NMR (400 MHz, CDCl₃) δ: 0.74 (3H, s),1.21 (6H, s), 1.57 (3H, br s), 2.30 (2H, m), 2.57 (1H, dd, J=13.3, 3.6Hz), 2.79 (1H, dd, J=11.6, 2.52 Hz), 2.85 (1H, dd, J=13.1, 4.44 Hz),4.48 (2H, m), 5.09 and 5.11 (1H and 1H, each s), 5.92 and 6.35 (1H and1H, each d, J=11.3 Hz); MS m/z (relative intensity):414 (M⁺, 90), 399(M-CH₃ ⁺, 17), 381 [M-CH₃—H₂O]⁺, 18), 363 ([M-CH₃-2×H₂O]⁺, 7), 285 (86),243 (35), 91 (100); exact mass calculated for C₂₇H₄₂O₃ ([M+Na]⁺) is437.3032, measured is 437.3026.

Schemes

Biological Activity of17(Z)-1α,25-Dihydroxy-17(20)-Dehydro-2-Methylene-19-Norvitamin D₃

The introduction of a methylene group to the 2-position, theintroduction of a double bond between the 17 and 20 positions, andorienting the side chain of 1α,25-dihydroxy-19-nor-vitamin D₃ in its Zconfiguration had little or no effect on binding to the full lengthrecombinant rat vitamin D receptor, as compared to1α,25-dihydroxyvitamin D₃. The compound VIT-III bound equally well tothe receptor as compared to the standard 1,25-(OH)₂D₃ (FIG. 1). It mightbe expected from these results that compound VIT-III would haveequivalent biological activity. Surprisingly, however, compound VIT-IIIis a highly selective analog with unique biological activity.

FIG. 5 shows that VIT-III has relatively high activity as compared tothat of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), the natural hormone, instimulating intestinal calcium transport.

FIG. 4 demonstrates that VIT-III has relatively high bone calciummobilization activity, as compared to 1,25(OH)₂D₃.

FIGS. 4-5 thus illustrate that VIT-III may be characterized as havingrelatively high, calcemic activity. Their preferential activity onintestinal calcium transport and calcium mobilizing activity allows thein vivo administration of these compounds for the treatment andprophylaxis of metabolic bone diseases. Because of their preferentialcalcemic activity on gut calcium transport and on bone, these compoundswould be preferred therapeutic agents for the treatment and prophylaxisof diseases such as osteoporosis, especially low bone turnoverosteoporosis, steroid induced osteoporosis, senile osteoporosis orpostmenopausal osteoporosis, as well as osteomalacia and renalosteodystrophy.

FIG. 2 illustrates that VIT-III is as potent as 1,25(OH)₂D₃ on HL-60cell differentiation, making it an excellent candidate for the treatmentof psoriasis and cancer, especially against leukemia, colon cancer,breast cancer, skin cancer and prostate cancer. In addition, due to itsrelatively high cell differentiation activity, this compound provides atherapeutic agent for the treatment of various skin conditions includingwrinkles, lack of adequate dermal hydration, i.e. dry skin, lack ofadequate skin firmness, i.e. slack skin, and insufficient sebumsecretion. Use of this compound thus not only results in moisturizing ofskin but also improves the barrier function of skin.

FIG. 3 illustrates that the compound VIT-III has about the sametranscriptional activity as 1α,25-dihydroxyvitamin D₃ in bone cells.This result, together with the cell differentiation activity of FIG. 2,suggests that VIT-III will be very effective in psoriasis because it hasdirect cellular activity in causing cell differentiation and insuppressing cell growth. These data also indicate that VIT-III may havesignificant activity as an anti-cancer agent, especially againstleukemia, colon cancer, breast cancer, skin cancer and prostate cancer,as well as a therapy for treating and/or preventing obesity.

The strong activity of VIT-III on HL-60 differentiation suggests it willbe active in suppressing growth of parathyroid glands and in thesuppression of the preproparathyroid gene. This analog having relativelyhigh calcemic activity is also expected to be useful as a therapy totreat hypoparathyroidism since it is effective to raise blood calciumlevels.

Experimental Methods

The compounds of the invention were prepared and studied using thefollowing methods.

Vitamin D Receptor Binding

Test Material

Protein Source

Full-length recombinant rat receptor was expressed in E. coli BL21 (DE3)Codon Plus RIL cells and purified to homogeneity using two differentcolumn chromatography systems. The first system was a nickel affinityresin that utilizes the C-terminal histidine tag on this protein. Theprotein that was eluted from this resin was further purified using ionexchange chromatography (S-Sepharose Fast Flow). Aliquots of thepurified protein were quick frozen in liquid nitrogen and stored at −80°C. until use. For use in binding assays, the protein was diluted inTEDK₅₀ (50 mM Tris, 1.5 mM EDTA, pH7.4, 5 mM DTT, 150 mM KCl) with 0.1%Chaps detergent. The receptor protein and ligand concentration wasoptimized such that no more than 20% of the added radiolabeled ligandwas bound to the receptor.

Study Drugs

Unlabeled ligands were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry (1,25(OH)₂D₃: molar extinctioncoefficient=18,200 and λ_(max)=265 nm). Radiolabeled ligand(³H-1,25(OH)₂D₃, ˜159 Ci/mmole) was added in ethanol at a finalconcentration of 1 nM.

Assay Conditions

Radiolabeled and unlabeled ligands were added to 100 mcl of the dilutedprotein at a final ethanol concentration of ≦10%, mixed and incubatedovernight on ice to reach binding equilibrium. The following day, 100mcl of hydroxylapatite slurry (50%) was added to each tube and mixed at10-minute intervals for 30 minutes. The hydroxylapaptite was collectedby centrifugation and then washed three times with Tris-EDTA buffer (50mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Titron X-100. After thefinal wash, the pellets were transferred to scintillation vialscontaining 4 ml of Biosafe II scintillation cocktail, mixed and placedin a scintillation counter. Total binding was determined from the tubescontaining only radiolabeled ligand.

HL-60 Differentiation

Test Material

Study Drugs

The study drugs were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry. Serial dilutions were prepared sothat a range of drug concentrations could be tested without changing thefinal concentration of ethanol (≦0.2%) present in the cell cultures.

Cells

Human promyelocytic leukemia (HL60) cells were grown in RPMI-1640 mediumcontaining 10% fetal bovine serum. The cells were incubated at 37° C. inthe presence of 5% CO₂.

Assay Conditions

HL60 cells were plated at 1.2×10⁵ cells/ml. Eighteen hours afterplating, cells in duplicate were treated with drug. Four days later, thecells were harvested and a nitro blue tetrazolium reduction assay wasperformed (Collins et al., 1979; J. Exp. Med. 149:969-974). Thepercentage of differentiated cells was determined by counting a total of200 cells and recording the number that contained intracellularblack-blue formazan deposits. Verification of differentiation tomonocytic cells was determined by measuring phagocytic activity (datanot shown).

In Vitro Transcription Assay

Transcription activity was measured in ROS 17/2.8 (bone) cells that werestably transfected with a 24-hydroxylase (24Ohase) gene promoterupstream of a luciferase reporter gene (Arbour et al., 1998). Cells weregiven a range of doses. Sixteen hours after dosing the cells wereharvested and luciferase activities were measured using a luminometer.RLU=relative luciferase units.

Antagonism was tested by adding a combination of 1,25(OH)₂D₃ and thecompound in the same well keeping the final ethanol concentration thesame.

Intestinal Calcium Transport and Bone Calcium Mobilization

Male, weanling Sprague-Dawley rats were placed on Diet 11 (Suda et al,J. Nutr. 100:1049, 1970) (0.47% Ca)+vitamins AEK for one week followedby Diet 11 (0.02% Ca)+vitamins AEK for 3 weeks. The rats were thenswitched to the same diet containing 0.47% Ca for one week followed bytwo weeks on the same diet containing 0.02% Ca. Dose administrationbegan during the last week on 0.02% calcium diet. Four consecutive ipdoses were given approximately 24 hours apart. Twenty-four hours afterthe last dose, blood was collected from the severed neck and theconcentration of serum calcium determined by atomic absorptionspectrometry as a measure of bone calcium mobilization. The first 10 cmof the intestine was also collected for intestinal calcium transportanalysis using the everted gut sac method.

Antagonism was tested by administering a combination of 1,25(OH)₂D₃ andthe compound to the animal simultaneously.

Interpretation of Data

VDR bindings, HL60 cell differentiation, and transcription activity.VIT-III (K_(i)=5.0×10⁻¹¹M) is equivalent to the natural hormone1α,25-dihydroxyvitamin D₃ (K_(i)=4.9×10⁻¹¹M) in its ability to competewith [³H]-1,25(OH)₂D₃ for binding to the full-length recombinant ratvitamin D receptor (FIG. 1). VIT-III is also significantly higher(EC₅₀=3.7×10⁻¹⁰M) in its ability (efficacy or potency) to promote HL60differentiation as compared to 1α,25-dihydroxyvitamin D₃(EC₅₀=1.3×10⁻⁹M) (See FIG. 2). Also, compound VIT-III (EC₅₀=4.4×10⁻¹²M)has greater transcriptional activity in bone cells than1α,25-dihydroxyvitamin D₃ (EC₅₀=2.9×10⁻¹⁰M) (See FIG. 3). These resultssuggest that VIT-III will be very effective in psoriasis because it hasdirect cellular activity in causing cell differentiation and insuppressing cell growth. These data also indicate that VIT-III will havesignificant activity as an anti-cancer agent, especially againstleukemia, colon cancer, breast cancer, skin cancer and prostate cancer,as well as against skin conditions such as dry skin (lack of dermalhydration), undue skin slackness (insufficient skin firmness),insufficient sebum secretion and wrinkles. It would also be expected tobe very active in suppressing secondary hyperparathyroidism.

Calcium mobilization from bone and intestinal calcium absorption invitamin D-deficient animals. Using vitamin D-deficient rats on a lowcalcium diet (0.02%), the activities of VIT-III and 1,25(OH)₂D₃ inintestine and bone were tested. As expected, the native hormone(1,25(OH)₂D₃) increased serum calcium levels at the dosage tested (FIG.4). FIG. 4 also shows that VIT-III has significantly greater activity inmobilizing calcium from bone than 1,25(OH)₂D₃. Administration of VIT-IIIat 260 pmol/day for 4 consecutive days resulted in significantmobilization of bone calcium, and increasing the amount of VIT-III to2340 pmol/day and then to 7020 pmol/day also increased the effect toprovide progressively increasing serum calcium levels well above theeffects seen with 1,25(OH)₂D₃.

Intestinal calcium transport was evaluated in the same groups of animalsusing the everted gut sac method (FIG. 5). These results show that thecompound VIT-III promotes a significant increase in intestinal calciumtransport activity when administered at 260 pmol/day, 2340 pmol/day or7020 pmol/day, as compared to 1,25(OH)₂D₃ at the 260 pmol/day dose.Thus, it may be concluded that VIT-III has significant intestinalcalcium transport activity at the tested doses.

These results illustrate that VIT-III is an excellent candidate fornumerous human therapies as described herein, and that it may beparticularly useful in a number of circumstances such as suppression ofsecondary hyperparathyroidism of renal osteodystrophy, autoimmunediseases, cancer, and psoriasis. VIT-III is an excellent candidate fortreating psoriasis because: (1) it has significant VDR binding,transcription activity and cellular differentiation activity; (2) it isdevoid of hypercalcemic liability unlike 1,25(OH)₂D₃; and (3) it iseasily synthesized. Since VIT-III has significant binding activity tothe vitamin D receptor, but has little ability to raise blood serumcalcium, it may also be particularly useful for the treatment ofsecondary hyperparathyroidism of renal osteodystrophy.

These data also indicate that the compound VIT-III of the invention maybe especially suited for treatment and prophylaxis of human disorderswhich are characterized by an imbalance in the immune system, e.g. inautoimmune diseases, including multiple sclerosis, lupus, diabetesmellitus, host versus graft rejection, and rejection of organtransplants; and additionally for the treatment of inflammatorydiseases, such as rheumatoid arthritis, asthma, and inflammatory boweldiseases such as celiac disease, ulcerative colitis and Crohn's disease.Acne, alopecia and hypertension are other conditions which may betreated with the compound VIT-III of the invention.

The compounds of the invention of formula I, and particularly formulaIa, are also useful in preventing or treating obesity, inhibitingadipocyte differentiations, inhibiting SCD-1 gene transcription, and/orreducing body fat in animal subjects. Therefore, in some embodiments, amethod of preventing or treating obesity, inhibiting adipocytedifferentiations, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal subject includes administering to the animalsubject, an effective amount of one or more of the compounds or apharmaceutical composition that includes one or more of the compounds offormula I. Administration of one or more of the compounds or thepharmaceutical compositions to the subject inhibits adipocytedifferentiation, inhibits gene transcription, and/or reduces body fat inthe animal subject. The animal may be a human, a domestic animal such asa dog or a cat, or an agricultural animal, especially those that providemeat for human consumption, such as fowl like chickens, turkeys,pheasant or quail, as well as bovine, ovine, caprine, or porcineanimals.

For prevention and/or treatment purposes, the compounds of thisinvention defined by formula I and Ia may be formulated forpharmaceutical applications as a solution in innocuous solvents, or asan emulsion, suspension or dispersion in suitable solvents or carriers,or as pills, tablets or capsules, together with solid carriers,according to conventional methods known in the art. Any suchformulations may also contain other pharmaceutically-acceptable andnon-toxic excipients such as stabilizers, anti-oxidants, binders,coloring agents or emulsifying or taste-modifying agents.

The compounds of formula I and particularly VIT-III of formula Ia, maybe administered orally, topically, parenterally, rectally, nasally,sublingually, or transdermally. The compound is advantageouslyadministered by injection or by intravenous infusion or suitable sterilesolutions, or in the form of liquid or solid doses via the alimentarycanal, or in the form of creams, ointments, patches, or similar vehiclessuitable for transdermal applications. A dose of from 0.01 μg to 1000 μgper day of the compounds I, particularly VIT-III, preferably from about0.1 μg to about 500 μg per day, is appropriate for prevention and/ortreatment purposes, such dose being adjusted according to the disease tobe treated, its severity and the response of the subject as is wellunderstood in the art. Since the compound exhibits specificity ofaction, each may be suitably administered alone, or together with gradeddoses of another active vitamin D compound—e.g. 1α-hydroxyvitamin D₂ orD₃, or 1α,25-dihydroxyvitamin D₃—in situations where different degreesof bone mineral mobilization and calcium transport stimulation is foundto be advantageous.

Compositions for use in the above-mentioned treatments comprise aneffective amount of the compounds I, particularly VIT-III, as defined bythe above formula I and Ia as the active ingredient, and a suitablecarrier. An effective amount of such compound for use in accordance withthis invention is from about 0.01 μg to about 1000 μg per gm ofcomposition, preferably from about 0.1 μg to about 500 μg per gram ofcomposition, and may be administered topically, transdermally, orally,rectally, nasally, sublingually or parenterally in dosages of from about0.01 μg/day to about 1000 μg/day, and preferably from about 0.1 μg/dayto about 500 μg/day.

The compounds I, particularly VIT-III, may be formulated as creams,lotions, ointments, topical patches, pills, capsules or tablets,suppositories, aerosols, or in liquid form as solutions, emulsions,dispersions, or suspensions in pharmaceutically innocuous and acceptablesolvent or oils, and such preparations may contain in addition otherpharmaceutically innocuous or beneficial components, such asstabilizers, antioxidants, emulsifiers, coloring agents, binders ortaste-modifying agents.

The compounds I, particularly VIT-III, may be advantageouslyadministered in amounts sufficient to effect the differentiation ofpromyelocytes to normal macrophages. Dosages as described above aresuitable, it being understood that the amounts given are to be adjustedin accordance with the severity of the disease, and the condition andresponse of the subject as is well understood in the art.

The formulations of the present invention comprise an active ingredientin association with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredients. The carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion.

Formulations for rectal administration may be in the form of asuppository incorporating the active ingredient and carrier such ascocoa butter, or in the form of an enema.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

Formulations suitable for topical administration include liquid orsemi-liquid preparations such as liniments, lotions, applicants,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes; or solutions or suspensions such as drops; or as sprays.

For nasal administration, inhalation of powder, self-propelling or sprayformulations, dispensed with a spray can, a nebulizer or an atomizer canbe used. The formulations, when dispensed, preferably have a particlesize in the range of 10 to 100μ.

The formulations may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.By the term “dosage unit” is meant a unitary, i.e. a single dose whichis capable of being administered to a patient as a physically andchemically stable unit dose comprising either the active ingredient assuch or a mixture of it with solid or liquid pharmaceutical diluents orcarriers.

1. A method of treating obesity of an animal, inhibiting adipocytedifferentiation, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal comprising administering to an animal in needthereof an effective amount of a 17(20)-dehydro vitamin D compoundhaving the formula:

where Y₁ and Y₂, which may be the same or different, are each selectedfrom the group consisting of hydrogen and a hydroxy-protecting group,where R₁₁ and R₁₂ are each hydrogen, where R₆ and R₇, which may be thesame or different, are each selected from the group consisting ofhydrogen, alkyl, hydroxyalkyl, fluoroalkyl, hydroxy and alkoxy, or R₆and R₇ when taken together may represent the group —(CH₂)_(x)— where xis an integer from 2 to 5, or R₆ and R₇ when taken together mayrepresent the group ═CR₈R₉ where R₈ and R₉, which may be the same ordifferent, are each selected from the group consisting of hydrogen,alkyl, hydroxyalkyl, fluoroalkyl, hydroxy and alkoxy, or when takentogether R₈ and R₉ may represent the group —(CH₂)_(x)— where x is aninteger from 2 to 5, and where the group R represents a side chainrepresented by the structure

where the side chain and 17-ene double bond is in the Z configurationand where Z in the above side chain structure is selected from Y, —OY,—CH₂OY, —C≡CY and —CH═CHY, where the double bond in the side chain mayhave the cis or trans geometry, and where Y is selected from hydrogen,—COR⁵ and a radical of the structure:

where m and n, independently, represent the integers from 0 to 5, whereR¹ is selected from hydrogen, deuterium, hydroxy, protected hydroxy,fluoro, trifluoromethyl, and C₁₋₅-alkyl, which may be straight chain orbranched and, optionally, bear a hydroxy or protected-hydroxysubstituent, and where each of R², R³, and R⁴, independently, isselected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyland C₁₋₅ alkyl, which may be straight-chain or branched, and optionally,bear a hydroxy or protected-hydroxy substituent, and where R¹ and R²,taken together, represent an oxo group, or an alkylidene group having ageneral formula C_(k)H_(2k)— where k is an integer, the group ═CR²R³, orthe group —(CH₂)_(p)—, where p is an integer from 2 to 5, and where R³and R⁴, taken together, represent an oxo group, or the group—(CH₂)_(q)—, where q is an integer from 2 to 5, and where R⁵ representshydrogen, hydroxy, protected hydroxy, or C₁₋₅ alkyl and wherein any ofthe CH— groups at positions 20, 22, or 23 in the side chain may bereplaced by a nitrogen atom, or where any of the groups —CH(CH₃)—,—(CH₂)_(m)—, —CR₁R₂— or —(CH₂)_(n)— at positions 20, 22, and 23,respectively, may be replaced by an oxygen or sulfur atom.
 2. The methodof claim 1 wherein the vitamin D compound is administered orally.
 3. Themethod of claim 1 wherein the vitamin D compound is administeredparenterally.
 4. The method of claim 1 wherein the vitamin D compound isadministered transdermally.
 5. The method of claim 1 wherein the vitaminD compound is administered in a dosage of from about 0.01 μg/date toabout 1000 μg/day.
 6. The method of claim 1 wherein the animal is ahuman.
 7. The method of claim 1 wherein the animal is a domestic animal.8. The method of claim 1 wherein the animal is an agricultural animal.9. A method of treating obesity of an animal, inhibiting adipocytedifferentiation, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal comprising administering to an animal in needthereof an effective amount of17(Z)-1α,25-dihydroxy-17(20)-dehydro-2-methylene-19-nor-vitamin D₃having the formula:


10. The method of claim 9 wherein17(Z)-1α,25-dihydroxy-17(20)-dehydro-2-methylene-19-nor-vitamin D₃ isadministered orally.
 11. The method of claim 9 wherein17(Z)-1α,25-dihydroxy-17(20)-dehydro-2-methylene-19-nor-vitamin D₃ isadministered parenterally.
 12. The method of claim 9 wherein17(Z)-1α,25-dihydroxy-17(20)-dehydro-2-methylene-19-nor-vitamin D₃ isadministered transdermally.
 13. The method of claim 9 wherein17(Z)-1α,25-dihydroxy-17(20)-dehydro-2-methylene-19-nor-vitamin D₃ isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.14. The method of claim 9 wherein the animal is a human.
 15. The methodof claim 9 wherein the animal is a domestic animal.
 16. The method ofclaim 9 wherein the animal is an agricultural animal.